For therapeutic applications, antibodies or antibody fragments must be of very high product quality. This requires, amongst others, homogeneity in structural terms. Moreover, the production costs are strongly influenced by difficulties encountered during the production process. Low yields or lack of homogeneity will impact the economics of the production process, and hence, the costs for the therapeutic, overall. For example, difficulties to separate structural variants of the desired antibody or antibody fragment will necessitate complex and costly purification strategies.
Amongst other requirements, therapeutic antibodies or their fragments must be properly folded. Protein folding is a spontaneous process leading to a uniquely folded structure depending on the given amino acid sequence. Cell surface and secreted proteins such as immunoglobulins often contain disulfide bonds (also referred to as disulfide bridges) that covalently link two cysteines and impart structural stability in the environment outside of the cell. An important event in the folding of these proteins is therefore the formation of these disulfide bonds. The number and position of the disulfide bridges will be determined by the number and location of suitable cysteine residues in the amino acid sequence of the antibody or antibody fragment.
The correct formation of all disulfide bridges is instrumental for proper folding and the stability of the obtained product. Proteins comprising disulfide bonds are oftentimes difficult to express recombinantly. For example, the expression of conventional immunoglobulins or their fragments, including Fab or scFv fragments, is problematic in terms of yield and functionality. For example, a conventional IgG molecule comprises a multitude of disulfide bonds both within single chains and between the four chains constituting the complete molecule. Early studies have already pointed to the difficulties in obtaining properly formed disulfide bridges for IgG molecules and have investigated various in vitro conditions (Litske & Dorrington J. Biol. Chem. 249: 5633, 1974).
In case one or more disulfide bridges are lacking in a conventional immunoglobulin (e.g. IgG, IgA, IgE, IgM), or a fragment derived therefrom, e.g. Fab, F(ab′)2 or scFv, functionality of the resulting product is typically compromised. Significant portions of the product obtained by recombinant expression will be non functional because of the missing disulfide bride(s), as widely reported in the art. Moreover, in the case of conventional antibodies or antibody fragments, such as Fab or scFv fragments, the formation of disulfide bridges has been reported to be rate limiting for the secretion of any product, in the first place.
It is known that e.g. formation of functional Fab, the heavy chain constant domains CH2 and CH3, or scFv is severely limited. For example, the amount of functional scFv may be entirely limited by correct disulfide formation (Ryabova et al., Nature Biotechn. 15: 79, 1997). The majority of the protein may form inactive aggregates, unless several folding helpers, including e.g. heavy chain binding protein (BiP) and protein disulfide isomerase (PDI) are overexpressed and act in an ATP dependent fashion (Mayer et al., J. Biol. Chem. 275: 29421, 2000; Lilie et al., J. Biol. Chem. 269: 14290, 1994; Vinci et al. J. Biol. Chem. 279: 15059, 2004; Mark et al., J. Biochem. 125: 328, 1999; Xu et al., Metabol. Engineer. 7: 269, 2005).
The limitation of obtaining adequate yields of functional product has been reported for conventional immunoglobulins and their fragments across a broad range of expression systems, including in vitro translation, E. coli, Saccharomyces cerevisiae, Chinese hamster ovary cells and baculovirus systems in insect cells or P. pastoris, amongst others (Ryabova et al., Nature Biotechnology 15: 79, 1997; Humphreys et al., FEBS Letters 380: 194, 1996; Shusta et al., Nature Biotech. 16: 773, 1998; Hsu et al., Protein Expr. & Purif. 7: 281, 1996; Mohan et al., Biotechnol. & Bioeng. 98: 611, 2007; Xu et al., Metabol. Engineer. 7: 269, 2005; Merk et al., J. Biochem. 125: 328, 1999; Whiteley et al., J. Biol. Chem. 272: 22556, 1997; Gasser et al., Biotechnol. Bioeng. 94: 353, 2006).
Thus the correct formation of canonical disulfide bridges is considered to be generally limiting to conventional antibody expression in microorganisms, including mammalian host cells.
In contrast to these difficulties observed with conventional four-chain antibodies or their fragments, including Fab and scFv, domain antibodies can be readily expressed and secreted in a correctly folded, fully functional form from hosts like E. coli or P. pastoris at a sufficient rate and level. Domain antibodies are characterized by formation of the antigen binding site by a single antibody domain, which does not require interaction with a further domain (e.g. in the form of VH/VL interaction) for antigen recognition. Production of Nanobodies, as one specific example of a domain antibody, in lower eukaryotic hosts such as Pichia pastoris has been extensively described in WO 94/25591.
The fact that fully functional domain antibodies can readily be produced in e.g. E. coli or yeast represents an important advantage of this immunoglobulin-format over conventional immunoglobulins. The production of domain antibodies in E. coli and yeast results in a good yield of functional product. The problems of obtaining sufficient amounts of functional product known from other immunoglobulin formats is hence unknown for domain antibodies.